Campylobacter bacteria are microorganisms that are pathogenic to humans as well as wild and domestic animals and that cause abortion and enteritis in animals and enteritis in humans. Campylobacter jejuni and Campylobacter coli are known to be causative bacteria of Campylobacter infection in humans. These bacteria are often referred to as food poisoning bacteria (Blaser, et al, Ann. Intern. Med., 91:179 (1979); Tauxe, R., American Society for Microbiology, Washington D.C. pg. 9 (1992)).
As of 2000, Campylobacter has been classified into 15 species and 9 subspecies. C. jejuni constitutes 95 to 99% of the bacteria that are isolated in human diarrhea cases, while other bacterial species, such as C. coli, constitutes only a few percent (Takahashi, M. et al, Infectious Diseases Weekly Report Japan, 3(6): 10 (2001)). However, the carriage rate of C. coli is extremely high in pigs. In recent years, Campylobacter infection has been on an increasing trend with increasing meat imports mainly from Southeast Asia. In particular, the infection from chicken-related food, whose consumption has been growing as a result of problems with beef such as BSE and O-157, has rapidly increased.
In addition, while Campylobacter fetus has been known as an abortion-causing bacteria in sheep and bovine, it has only recently been reported to be involved in abortion and premature delivery in humans as well. C. fetus infection, resulting from eating raw liver or beef contaminated with C. fetus, is associated with symptoms such as sepsis and meningitis. The primary source of Campylobacter infection in humans is chicken, which carries the bacteria at high densities in the intestinal tract (Simon, M. S. et al., 2003. Campylobacter infection. Diseases of Poultry, Iowa State Press, 615-630).
Campylobacter bacteria are generally distributed at a high density in the digestive tract of animals, such as bovine, sheep, pig, and chicken, and thus recognized as causative bacteria of zoonosis. Most campylobacteriosis is thought to be caused by chicken. Infection may arise through direct contact with the above animals or their excrement, or through intake of or during processing of food, drinking water, milk, and such contaminated with the excrement. Furthermore, infection cases in facilities such as newborn nurseries have also been reported (Japanese Journal of Pediatric Medicine, 29:1219-1222 (1997)).
Campylobacteriosis has a long incubation period, ranging 3 to 7 days. It is characterized by gastroenteritis symptoms, such as diarrhea (sometimes, bloody mucous diarrhea), abdominal pain, fever, nausea, vomiting, headache, chills, and feebleness. Although the lethality is low, newborn babies may develop systemic infection, such as sepsis and meningitis. In most cases, recovery takes several days to about one week. The general prognosis has a favorable course except in some immunodeficiency patients. However, it has been reported in recent years that patients may develop Guillain-Barre syndrome or Fischer syndrome, which are autoimmune diseases, after campylobacteriosis. The cases developed following campylobacteriosis generally tend to become severe, and the remission rate after one year of the onset is only about 60%.
Chemotherapy using antibiotics is performed for severe conditions or cases complicated by sepsis. The first choice drug is a macrolide, such as erythromycin. Due to natural resistance, cephem antibiotics are not expected to have therapeutic effects. Meanwhile, the increase in the number of bacteria resistant to new quinolone antibiotics has become a problem in recent years. Rapid identification of causative microorganisms after infection is important to conduct an appropriate treatment for campylobacteriosis and to prevent the expansion of infection by revealing the infection route. However, it is difficult to diagnose campylobacteriosis based on clinical symptoms alone, much less to identify Campylobacter and its species.
Campylobacter bacteria are microaerophiles. A culture of the bacteria requires a special medium such as Skirrow's medium, and a special apparatus (anaerobic jar or the like) to maintain the oxygen concentration at 3 to 10% for the absolute microaerophilic condition. In addition, the culture is time-consuming (2 to 3 days) as compared with other bacteria. Thus, it is difficult to achieve and maintain an isolation culture of Campylobacter bacteria. Furthermore, since Campylobacter bacteria easily die in the air, they must be tested within 2 to 3 hours after sample collection. Furthermore, since the incubation period of campylobacteriosis is long (3 to 7 days), the bacteria often cannot be isolated when bacterial identification in any foods concerned is carried out after the onset of the symptoms. Furthermore, Campylobacter bacteria have very strong infectivity, and have been reported to establish infection with only several hundreds of cells. Thus, it is extremely difficult to identify the source of infection.
One method of discriminative diagnosis between C. jejuni and C. coli involves testing hippurate hydrolysis. Specifically, the method is based on the fact that C. jejuni has the ability to hydrolyze hippurate while C. coli does not. However, this method is not exact because there some hippurate-negative C. jejuni species are known in the art (Totten, et al, J. Clin. Microbiol., 25: 1747 (1987)). Thus, the presence of Campylobacter bacteria can be confirmed only by estimating the presence of the bacteria from food intake history and symptoms, and by examining morphological and biological features of bacteria from colonies obtained by feces culture, which requires several days.
Thus, attempts have been made to identify Campylobacter bacteria and detect its toxin genes using, as rapid diagnostic methods that don't require cultivation, genetic diagnostic methods which utilize a DNA probe method or a PCR method using oligonucleotides. For example, the gene encoding rRNA has been generally used as a probe for Campylobacter bacteria (Japanese Patent Application Kokai Publication No. (JP-A) S62-228096 (unexamined, published Japanese patent application)). The sequences of Campylobacter rRNA genes have already been published (Romaniuk, P. J. et al, J. Bacteriol., 169: 2173 (1987)). In addition, nucleic acid fragments for detecting Campylobacter bacteria are also known (JP-A H2-84200; JP-A H2-154700; JP-A H3-112498; JP-A H6-90795; JP-A H6-90796). However, while these sequences may be used to detect C. jejuni and/or C. coli, they are not adequate to detect other Campylobacter bacteria. Furthermore, the current level of specificity is not sufficient.
A method for identifying C. jejuni by PCR, using oligonucleotides selected from the fla A gene of C. coli VC167, has also been reported (Oyofo, et al, J. Clin. Microbiol., 30: 2613 (1992)). Furthermore, the use of oligonucleotide primers to amplify a target sequence of superoxide dismutases of C. jejuni and C. coli has been reported in the literature (Romaniuk, P. J. et al, J. Bacteriol., 169: 2173 (1987)). However, these methods are incapable of discriminating between C. jejuni and C. coli. 
Meanwhile, pathogenic factors of Campylobacter are being studied actively. Various factors, such as cell invasiveness, flagellin, and cholera toxin-like enterotoxin, have been reported as pathogenic factors of Campylobacter bacteria (Mizuno, K. et al, Microbios., 78: 215 (1994); Suzuki, S. et al, FEMS Immunol. Med. Microbiol., 8: 207 (1994)). Recently, cytolethal distending toxin (CDT) was discovered as a toxic factor from C. jejuni (Pickett, C. et al. Infect. Immun., 64: 2070 (1996)), and its relevance to the pathogenicity has attracted attention. For example, diarrheagenicity of the toxin has been reported in an animal model using recombinant E. coli that produces CDT of Shiga's bacillus (Shigella dysenteriae) (Infect. Immun., 65: 428-433 (1997)).
CDT is a holotoxin composed of three subunits, called cdtA, cdtB, and cdtC, which are encoded by genes arranged in tandem. The active center of the toxin is in the cdtB subunit having type I deoxyribonuclease-like activity, while the cdtA and cdtC subunits are thought to be involved in the adhesion to target cells. When the holotoxin acts on cells, the cells are distended, i.e. swollen, and finally killed. The toxin is thus named “cytolethal distending toxin”.
The molecular mechanism is believed to be as follows. The cdtB subunit that constitutes the active center of the toxin translocates into a cell nucleus, and introduces nicks into chromosomal DNA by its type I deoxyribonuclease activity, thereby inducing DNA-damage response. The cell then arrests the cell cycle at G2/M phase to activate the gene repair system, and is then distended and killed (Science, 290: 354-357 (2000)). Furthermore, CDT has been found to act on a broad range of cells, including epithelial cells and immune cells. In particular, CDT is believed to act on human lymphocytes and induce apoptosis in them, which allows easy escape from immunity (J. Biol. Chem., 276: 5296-5302 (2001)).
As described above, CDT has a unique molecular mechanism that is not found in the other toxins previously known. To date, the complete nucleotide sequence of CDT among Campylobacter bacteria has been determined for only C. jejuni (Pickett, C. et al. Infect. Immun., 64: 2070 (1996)).